Study reveals promising information for developing an alternative to antibiotics

The analysed endolysins are activated by switching from a tensed, stretched state (left) to a relaxed state (right). Credit: EMBL/Rob Meijers.

In a nutshell:

How bacteriophages demolish C. difficile’s cell wall

Discovered structural switch that activates the enzymes involved

Could help engineer viruses to target specific bacteria

Research published today in PLOS Pathogens reveals how viruses called bacteriophages destroy the bacterium Clostridium difficile (C. diff), which is becoming a serious problem in hospitals and healthcare institutes, due to its resistance to antibiotics. The study, by scientists at the European Molecular Biology Laboratory (EMBL) in Hamburg, Germany, could help bring about a new way of fighting this and other bacteria.

“Our findings will help us to engineer effective, specific bacteriophages, not just for C. diff infections, but for a wide range of bacteria related to human health, agriculture and the food industry,” says Rob Meijers from EMBL, who led the work.

C. diff infections, which can be fatal, are currently very difficult to treat, as the bacterium is particularly unresponsive to many antibiotics. A possible solution would be not to use antibiotics, but instead employ bacteriophages – viruses which infect only bacteria. Scientists know that these viruses hijack a bacterium’s DNA-reading machinery and use it to create many new bacteriophages. These then start demolishing the bacterium’s cell wall. Once its wall begins to break down, the bacterial cell can no longer withstand its own internal pressure and explodes. The newly formed viruses burst out to find new hosts and the bacterium is destroyed in the process.

To harness the power of bacteriophages and develop effective therapies against bacteria like C.diff, scientists need to know exactly how these viruses destroy bacterial cell walls. The viruses’ demolition machines, endolysins, are known, but just how these enzymes are activated was unclear – until now.

“These enzymes appear to switch from a tense, elongated shape, where a pair of endolysins are joined together, to a relaxed state where the two endolysins lie side-by-side,” explains Matthew Dunne who carried out the work. “The switch from one conformation to the other releases the active enzyme, which then begins to degrade the cell wall.”

Meijers and collaborators discovered the switch from ‘standby’ to ‘demolition’ mode by determining endolysins’ 3-dimensional structure, using X-ray crystallography and small angle X-ray scattering (SAXS) at the Deutsches Elektronen-Synchrotron (DESY). They compared the structures of endolysins from two different bacteriophages, which target different kinds of Clostridium bacteria:one infects C. diff, the other destroys a Clostridium species that causes defects in fermenting cheese.

Remarkably, the scientists found that the two endolysins share this common activation mechanism, despite being taken from different species of Clostridium. This, the team concludes, is an indicator that the switch between tense and relaxed enzymes is likely a widespread tactic, and could therefore be used to turn other viruses into allies in the fight against other antibiotic-resistant bacteria.

The work was performed in collaboration with Arjan Narbad’s lab at the Institute of Food Research in Norwich, UK, who tested how engineering mutations in the endolysins affected their ability to tear down the bacterial cell wall.

Article Abstract

The bacteriophage ΦCD27 is capable of lysing Clostridium difficile, a pathogenic bacterium that is a major cause for nosocomial infection. A recombinant CD27L endolysin lyses C. difficile in vitro, and represents a promising alternative as a bactericide. To better understand the lysis mechanism, we have determined the crystal structure of an autoproteolytic fragment of the CD27L endolysin. The structure covers the C-terminal domain of the endolysin, and represents a novel fold that is identified in a number of lysins that target Clostridia bacteria. The structure indicates endolysin cleavage occurs at the stem of the linker connecting the catalytic domain with the C-terminal domain. We also solved the crystal structure of the C-terminal domain of a slow cleaving mutant of the CTP1L endolysin that targets C. tyrobutyricum. Two distinct dimerization modes are observed in the crystal structures for both endolysins, despite a sequence identity of only 22 % between the domains. The dimers are validated to be present for the full length protein in solution by right angle light scattering, small angle X-ray scattering and cross-linking experiments using the cross-linking amino acid p- benzoyl-L-phenylalanine (pBpa). Mutagenesis on residues contributing to the dimer interfaces indicates that there is a link between the dimerization modes and the autocleavage mechanism. We show that for the CTP1L endolysin, there is a reduction in lysis efficiency that is proportional to the cleavage efficiency. We propose a model for endolysin triggering, where the extended dimer presents the inactive state, and a switch to the side-by-side dimer triggers the cleavage of the C-terminal domain. This leads to the release of the catalytic portion of the endolysin, enabling the efficient digestion of the bacterial cell wall.

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